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Abstract (250 words).Antibody subclasses exhibit extensive polymorphisms (allotypes) that could potentially impact the quality of HIV-vaccine induced B cell responses. Allotypes of IgG1, the most abundant serum antibody, have been shown to display altered functional properties in regard to serum half-life, Fc-receptor binding and FcRn-mediated mucosal transcytosis. To investigate the potential link between allotypic IgG1-variants and vaccine-generated humoral responses in a cohort of 14 HIV vaccine recipients, we developed a novel protocol for rapid IgG1-allotyping.We combined PCR and ELISA assays in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1) of trial participants, using human plasma and RNA isolated from PBMC. The IgG1-allotype distribution of our participants mirrored previously reported results for caucasoid populations. We observed elevated levels of HIV gp140-specific IgG1 and decreased IgG2 levels associated with the G1m1-allele, in contrast to G1m3 carriers. These data suggest that vaccinees homozygous for G1m1 are predisposed to develop elevated Ag-specific IgG1:IgG2 ratios compared to G1m3-carriers. This elevated IgG1:IgG2 ratio was further associated with higher FcɣR-dimer engagement, a surrogate for potential ADCC and ADCP function.Although preliminary, these results suggest that IgG1 allotype may have a significant impact on IgG subclass distribution in response to vaccination and associated Fc-mediated effector functions. These results have important implications for ongoing HIV vaccine efficacy studies predicated on engagement of FcɣR mediated cellular functions including ADCC and ADCP, and warrant further investigation. Our novel allotyping protocol provides new tools to determine the potential impact of IgG1 allotypes on vaccine efficacy.□1 IntroductionAntibodies are generally accepted to contribute to vaccine induced protection against many infectious diseases including HIV (Plotkin, 2010). However, the involvement of different antibody classes or subclasses is less clear, where their different structural properties affect functional immunity. While antibody binding fragments (Fabs) are critical to determining binding specificity and neutralisation, the Fc domain is the primary determinant for a wide spectrum of immunological functions mediated by the engagement of Fc-gamma receptors (FcγR) on a range of effector cells. These functions are regulated during an immune response through Ig subclass composition tailoring the selective interaction with FcγR on effector immune populations. Indeed, Ig subclass composition influences a wide range of Fc-mediated effector functions including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) (Vidarsson et al., 2014). In this respect Fc-mediated effector functions likely augment the potency of broadly neutralizing antibodies (Bournazos et al., 2014) and are critical to the function of non-neutralizing antibodies. The observed modest efficacy of the RV144 HIV vaccine trial showing vaccine elicited protection in the absence of neutralizing antibodies (Rerks-Ngarm et al., 2009) has driven an intense interest in the role of FcγR effector functions in protection and control of HIV infection (Excler et al., 2014; Tomaras and Plotkin, 2017). Here the antibody subclass distribution is likely to play a critical role where IgG1/IgG3 interact efficiently with most FcγR, while IgG2/IgG4 show reduced affinity for many FcγR. Indeed, divergent antibody subclass profiles have been associated with variable antibody effector functions among HIV-1 controllers, where levels of HIV-specific IgG1/3 were the major distinguishing factor (Lai et al., 2014), while IgG2/IgG4 HIV specific antibodies were associated with poorer overall antibody activity (Ackerman et al., 2016). In this respect, inter-individual variation in the antibody subclass response profiles to HIV-1 infection and/or vaccination provides significant challenges in the development of a globally effective vaccine. In addition to sequence diversity of variable Fab domains and isotypic variation, IgG-subclass antibodies have been shown to exhibit polymorphic epitopes (IgG-allotypes), which can differ between individuals and ethnic groups (Vidarsson et al., 2014). Inherited in a codominant Mendelian fashion, IgG-heavy chain allotypes are designated as natural genetic marker (Gm) together with the antibody subclass (e.g. G1m) and the allotype number (e.g. G1m3 or G1m1) (Jefferis and Lefranc, 2009). So far, a total of 4 G1m human allotypes:G1m17, G1m3, G1m1 and G1m2; two G1m alloallotypes: G1m27 and G1m28; and two G1m isoallotypes: nG1m17 and nG1m1 have been identified via serological typing (Lefranc and Lefranc, 2012). These define 7 G1m alleles: G1m17,1; G1m3; G1m17,1,27; G1m17,1,28; G1m17,1,27,28; G1m17,1,2; and G1m3,1; where the G1m1 allotype is common to all alleles except G1m3. The prevalence of these alleles broadly differs according to European, African or Asian ancestry. Most Gm allotypes are located in the Fc-region (CH2 or CH3) of antibodies, with the exception of G1m3 which is linked to amino acid changes in the CH1-region: expressing Arg rather than Lys at position 120 (Lefranc and Lefranc, 2012; Vidarsson et al., 2014). G1m3 also expresses unique amino acids at positions 356 (Glu) and 358 (Met) in CH3 as opposed to Asp/Leu common to all G1m1 allotypes. While allotypes are encoded by one given immunoglobulin gene, some amino acid variations can be found in antibody chains of other isotypes (isoallotypes). For example, the amino acid residue Arg120, which corresponds to G1m3, is also found in antibodies belonging to the IGHG3 and IGHG4 allele family (Lefranc and Lefranc, 2012; Ternant et al., 2016). Prior work has linked Gm allotypes in the immunoglobulin constant heavy G chain (IGHG) to augmented antibody responses against certain diseases (Ambrosino et al., 1985; Pandey et al., 2008; Migot-Nabias et al., 2011; Oxelius and Pandey, 2013). For example, IgG antibody responses against the hepatitis C virus (HCV) envelope proteins E1E2 in a cohort of infected subjects with GM 1,17 5,13 and KM 1 phenotypes exhibit four-fold higher levels of E1E2-specific antibodies (Pandey et al., 2008). Another study showed that IgG1 antibody levels to malaria vaccine-antigens were significantly higher in subjects with the GM 3 23 5,13,14 phenotype when compared to subjects lacking this phenotype (Pandey et al., 2010).Similar trends have been reported for IgG-subclass and specificity profiles in a cohort of elite HIV-1 controllers where HIV-specific IgG1 levels correlated with Fc-dependent effector functions and total plasma IgG1. Subsequently, it was argued that Gm allotypes could be responsible for variations in IgG1-concentrations and it was suggested that future studies should incorporate Gm allotyping protocols to account for this possibility (Lai et al., 2014). Furthermore, previous studies have demonstrated the involvement of Gm alleles in ADCC of cancer cells (Pandey et al., 2013; Pandey and Namboodiri, 2014b). Taken together these studies indicate that Gm allotypes impact on antibody functionality (Pandey, 2014) and provided a strong rationale for investigating the impact of IgG1-allotypes on the magnitude and functionality of vaccine-induced IgG-subclasses responses in the context of an HIV-vaccine trial. Traditionally the Gm phenotype have been determined via haemagglutination inhibition assays (HAI) using anti-Rh IgG antibodies of known allotypy, and polyclonal IgG of a selected allotype-specificity (e.g. anti-human G1m3). However, access to such reagents can be rate limiting and the approach is less amenable to high volume screening. Given the dominant role of IgG1 responses to HIV-1 envelope immunogens (Banerjee et al., 2010) we sought to determine the impact of IgG1-allotypy on the magnitude of induced responses. Here, we combined PCR and ELISA assays in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1) of clinical trial participants, using human plasma and RNA isolated from PBMC. Subsequently, the distribution of IgG1-allotypes formed the framework for assessing the effect of IgG1-allotypes on the magnitude and functionality of vaccine-induced antibody responses. Understanding how IgG1 allotypy influences IgG subclass distribution in response to vaccination may prove an important consideration in the design and evaluation of vaccines strategies across ethnic groups.2 Materials and Methods2.1 HIV vaccine trialThis study mainly builds upon findings from the previously published X001 clinical trial (Kratochvil et al., 2017). In brief, a recombinant clade C HIV-1 envelope gp140 protein (CN54gp140) produced by Polymun Scientific (Klosterneuburg, Austria) to GMP specification, which has been reported to be immunogenic in a number of preclinical and clinical studies (Lewis et al., 2011; Cosgrove et al., 2016), was used. The vaccine antigen CN54gp140 was administered intramuscularly into the deltoid muscle of the upper arm at a dosage of 100 µg CN54gp140 formulated with 5 µg GLA-AF (Glucopyranosyl Lipid A - Aqueous Formulation, Infectious Disease Research Institute, Seattle, USA (Arias et al., 2012)) in a total volume of 0.4 ml at week 0, 4 and 8 with a boost inoculation with the same material at either month 6 or 12. The trial was performed at the NIHR/Wellcome Trust Imperial Clinical Research Facility, Imperial College, London. The trial population was predominantly Caucasian, although subject ethnicity was not recorded as part of the trial2.2 Ethics statementThe clinical trials generating serum and PBMC samples were conducted in compliance with UK Clinical Trial Regulations and any amendments, which include compliance with the principles of Good Clinical Practice (GCP), and the study abided by the principles of the Declaration of Helsinki. All volunteers provided written informed consent to participate in the trials on the basis of appropriate information and with adequate time to consider the information and discuss the trial with the Principal Investigators or their delegate. The trial proposal, the trial-specific information provided to volunteers, the consent form and substantial protocol amendments (if applicable) were reviewed by a recognized Research Ethics Committee (REC) and by the Medicines and Healthcare products Regulatory Authority (MHRA). All volunteers were made aware that they were free to withdraw without obligation at any time and that such an action would not adversely affect any aspect of their medical care or legal rights.2.3 PBMC isolationPBMC were isolated using density gradient separation from heparinized whole blood, used fresh (within 8 hours of blood collection) or frozen in a mixture of fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA) and DMSO at a 90:10 ratio using a Kryo 560-16 rate controlled freezer (Planer, Sunbury-On-Thames, UK). PBMC were stored in vapour phase liquid nitrogen.2.4 Determining the IgG1-allotype identityHuman RNA was isolated from PBMC with the RNeasy Mini Kit for RNA (Qiagen, UK) and transcribed into cDNA via oligo(dT)18 primers, using the maxima first strand cDNA synthesis kit (Cat: K1672, Thermo Scientific, UK). Subsequently, the template cDNA was used for a primary PCR for both G1m3 and G1m1 allotyping. While the primary PCR is sufficient to account for G1m1,17 alleles, G1m3-allotyping required a secondary PCR to take account of (and exclude) isoallotypes that can be present in IgG3 and IgG4 regions. Primers and PCR programs are detailed in the supplementary section (Tables S1 to S5). 2.5 Agarose gelsDNA products were separated by 1.2% agarose gel electrophoresis (100 V, 1 h) in 1x Tris Acetate EDTA (TAE) buffer. The agarose gel was stained with SYBR Safe (1 in 20,000), a suitable DNA ladder was loaded and 5x loading dye was added to each sample prior to loading the gel. DNA was visualised on a transilluminator.2.6 ELISA protocol for IgG1-allotyping2.6.1 G1m3-ELISASerum antibodies from clinical trial participants were assessed for the presence of the IgG1-allotype G1m3 via a novel ELISA protocol adapted from a previously published ELISA platform ((Kratochvil et al., 2017)). In brief, 96-well high binding MaxiSorp plates (Nunc) were coated with 100 µl/well anti-G1m3 (Cat: I5385-.2ML, Sigma, UK), at a 1:5,000 dilution in PBS, overnight at 4˚C. As reference material, standard human immunoglobulins, which were captured with a combination of anti-human kappa and lambda light chain specific mouse antibodies, were used. These capture antibodies were coated onto the plates overnight at 4˚C and coated plates were washed four times with PBS-T before blocking with PBS supplemented with 1%BSA and 0.05% Tween-20. Following further washing, diluted serum samples were added to the pre-coated wells (generally between 1:10,000 to 1,000,000) and titrations of immunoglobulin standards were added to the kappa/lambda capture antibody coated wells at 50 µl/well and incubated for 1 hour at 37˚C. Plates were washed four times prior to the addition of anti-human IgG-HRP and incubated for 1 hour at 37˚C. Plates were washed four times and developed with 50 µl/well of KPL SureBlue TMB substrate (Insight Biotechnology, UK). The reaction was stopped after 5 min by adding 50 µl/well of 1 M H2SO4, and the absorbance read at 450 nm on a KC4 spectrophotometer.2.6.2 G1m1 ELISAFor the G1m1 ELISA the commercially available detection antibody anti-IgG1-Hinge-HRP (Cat: 9052-08, Southern Biotech) was used at a 1:5,000 dilution. Except for the detection antibody, the G1m1-ELISA is identical to the anti-IgG1-ELISA protocol previously published (Kratochvil et al., 2017). This antibody does not bind to G1m3 allele, but does show cross reactivity with G1m3,1 allele prevalent in those with Asian ancestry, suggestive of recognition of the common G1m1 allotype. 2.7 Customized multiplex dimer assay for the assessment of FcR-binding.A customized multivariate multiplex assay was developed using a panel of gp140 antigens (Clade C: CN54, 1086, Clade A: UG37, Clade D: UG21–NIH AIDS Reagents) coupled to magnetic fluorescent multiplex beads (Bio-Rad, AU) as described previously (Brown et al., 2012). Biotinylated dimeric Fc-gamma-Receptors (FcγRIIa-H131, FcγRIIIa-V158) were produced as previously described (Wines et al., 2016). The method has been previously published ((Kratochvil et al., 2017)).2.8 Statistical methodsStatistical analysis was carried out using Prism 7.0a (GraphPad, CA, USA) or the R software (R3.3.2) for statistical programming (Team, 2008). 3 Results3.1 Cross-validation of a novel ELISA protocol for rapid IgG1-allotyping against a PCR protocolWe wished to assess whether IgG-allotypes might be linked to differences in the IgG-subclass profile of Ag-specific antibody responses generated in the context of HIV-1 vaccination (Pandey and Namboodiri, 2014a; Webster et al., 2016). To pursue this, we developed novel PCR (Supplementary Material, Fig S1) and ELISA protocols and combined them in a dual approach to determine the IgG1 allotype identity (G1m3 and/or G1m1) of clinical trial participants, using both human serum and mRNA. The optimized protocol was applied to determine the IgG1 allotype identity of 14 clinical trial participants from an HIV vaccine study utilizing a Clade C gp140 envelope protein (Fig 1) (Kratochvil et al., 2017). IgG1-allotyping X001 study participants revealed that 3/14 (21%) were homozygous for G1m1 and 6/14 (43%) homozygous for G1m3, with 5/14 (36%) carrying both alleles (heterozygous). The IgG1 allotype distribution mirrors previously reported most frequent alleles representative of Caucasian populations (G1m3; G1m 17, 1; and G1m17,1,2) and the concurring results demonstrate the interchangeability of the PCR and ELISA protocol for IgG1-allotyping. The clear cross-validation of both assays formats, allowed for the determination of the IgG1-allotype distribution in two additional clinical trials (Cosgrove et al., 2016; Joseph et al., 2017), from which only serum samples were available (Supplementary Material, Fig S2). 3.2 Implications of IgG1-allotypes for the analysis of HIV-vaccine induce IgG-subclass responses.The IgG1-allotype identities determined for the X001 study (Kratochvil et al., 2017) provided a distinctive framework for the re-analysis of HIV vaccine-induced IgG-subclass profiles (Fig 2). Most participants, homo- or heterozygous for G1m1, exhibited a trend for higher Ag-specific IgG1 concentrations when compared to homozygous G1m3-carriers (Fig 2A), while the trend was reversed for Ag-specific IgG2 responses (Fig 2B). By contrast there were no identified differences in Ag-specific IgG3 and IgG4 responses according to G1m1 allotype (data not shown). Although the X001 study (Kratochvil et al., 2017) was not powered sufficiently to demonstrate a statistical significance between homologous G1m1- and G1m3-allele carriers, the apparent differences in Ag-specific IgG1/ IgG2 levels warranted further investigation. All participants with at least one G1m1-allele were then analysed as a single group and directly compared with homozygous G1m3-carriers. The subsequent AUC-analysis helped to further unravel the effect of G1m1-allotypy on the magnitude of Ag-specific IgG1/IgG2 responses by revealing trends and potential differences (p=0.0823) between G1m1-carriers and homologous G1m3-carriers (Fig 2C). Furthermore, the highest Ag-specific IgG1 responses that occurred 14 days after the second IM and 14 days after the fourth IM were in G1m1-carriers (Fig 2A).3.3 Association of IgG1-allotypes and differences in ratios of antigen-specific IgG1/IgG2-levels following serial immunizations with CN54gp140.Following these initial observations, additional serum samples from the related MUCOVAC2 trial (EudraCT 2010-019103-27 (Cosgrove et al., 2016)) were made available for IgG1-allotyping and measurement of IgG1-levels (Fig 4) using our established ELISA protocols. MUCOVAC2 is a predecessor study to X001 and was designed to establish the optimal route and dosage of immunization with the candidate HIV-1 clade C CN54gp140 envelope glycoprotein vaccine (Cosgrove et al., 2016). For two participants the timing (3rd IM, week8), immunogen (CN54gp140) and dose (100ug) in the MUCOVAC2 trial was identical to week 8 (3rd IM) in the X001 study. Thus, it was possible to pool data from the two clinical trials for this selected time point, allowing for a follow-up analysis of the differences in Ag-specific IgG1/IgG2 ratios mediated by G1m1 (Fig 3A: homozygous, n=4; Fig 3B: homo- and/ or heterozygous, n=6) and G1m3 (homozygous, n=7).Following the 3-immunisation priming phase, volunteers homozygous for G1m1 (n=4) had 5-fold higher Ag-specific IgG1/IgG2 ratios in comparison to homozygous G1m3-carriers (n=7, p=0.0242, Fig 3A). However, no significant differences in Ag-specific IgG1/ IgG2 ratios were observed when comparing both hetero- and homozygous G1m1-carriers with homozygous G1m3-carriers when using this larger data set (p=0.1807, Fig 3B).3.4 Correlational relationships between FcɣR-binding (FcɣRIIa/ FcɣRIIIa) and Ag-specific IgG1 levels were determined with respect to different combinations of G1m1 and/or G1m3 alleles.Despite statistical limitations in study power, evidence was found to suggest that Ag-specific IgG1/IgG2 levels varied according to the IgG1-allotype of the HIV-vaccine recipients. To further elucidate the role of allotypic variations in antibody responses, the impact of IgG1-allotypes on the magnitude of Fc-mediated functions was investigated. Investigating these observations in the context of an HIV-vaccine trial was facilitated by the combination of a novel assay, using FcγR-ectodomains for probing Fc-mediated functions (Wines et al., 2016; Stachowiak et al., Spring 2010), and the novel IgG1-allotyping protocol. Correlational relationships between FcɣR-binding (FcɣRIIa/ FcɣRIIIa) and Ag-specific IgG1 levels were determined with respect to different combinations of G1m1 and/or G1m3 alleles in X001 study participants (Fig 4).The engagement of FcɣRIIa/ FcɣRIIIa dimers via CN54gp140-sepcifc serum antibodies correlated significantly (p<0.0001) with Ag-specific IgG1 levels, irrespective of the IgG1-allotype combinations. Furthermore, it is notable that the maximum of FcR-binding and Ag-specific IgG1 levels were higher in individuals that are homo- or heterozygous for the G1m1-allele (Fig 4A and C) when compared to homozygous G1m3-carriers (Fig 4B). □DiscussionA novel allotyping protocol was developed and employed to determine the distribution of IgG1-allotypes in HIV vaccine studies (X001 and MUCOVAC1 clinical trials). The allotype distributions were found to mirror those previously reported for Caucasian populations, in which the G1m3-allele is known to be predominant (Johnson et al., 1977; Magdelaine-Beuzelin et al., 2009; Lefranc and Lefranc, 2012; Oxelius and Pandey, 2013). Interestingly, the frequency of donors homozygous for G1m3 (45.4%) reported for a larger study (570 community blood donors) coincided with the G1m3-distribution determined for the X001 study (Webster et al., 2016). These results provided a framework for regrouping and re-analyzing the X001 Ag-specific IgG-subclass data in the context of IgG1-allotypes. Initial data analysis of individual Ag-specific IgG1 responses in study participants suggested a link between G1m1-carriers (homozygous or heterozygous) and elevated Ag-specific IgG1 responses when compared to homozygous G1m3-carriers. Thus, Ag-specific IgG1 concentrations from both homo- and heterozygous G1m1 carriers were grouped and compared with homozygous G1m3-carriers, revealing differences in Ag-specific IgG1 levels between the two study groups. The results align well with a previous study, in which different lower serum IgG and IgG-subclass levels were associated with G1m3, G3m5 allotypes in a study population of 157 Caucasian blood donors (Oxelius, 1993) and mirror the observations made by Lai et al. (Lai et al., 2014). In contrast to this finding, HIV-1 vaccine recipients with at least one G1m1 allele appeared to have lower Ag-specific IgG2 levels in comparison to homozygous G1m3-carriers. Subsequently, the Ag-specific IgG1:IgG2-ratio was compared for time- and dose-matched samples from two additional clinical HIV-vaccine trials. The data suggest that vaccinees homozygous for G1m1 have elevated Ag-specific IgG1:IgG2 ratios compared to G1m3-carriers. Homozygosity for the G1m3-allele is strongly linked to the G2m23-allele in Northern Europe (Jefferis and Lefranc, 2009). Previous studies have suggested that individuals homozygous for this allotype (G2m23) exhibit higher serum IgG2 titers and decreased anti-polysaccaride IgG2 responses than those homozygous for G2m(..), heterozygotes having intermediate levels (Ambrosino et al., 1985; Granoff and Holmes, 1992; Jefferis and Lefranc, 2009; Pandey, 2014). Further work would be required to determine any additional contribution of G2m23 to the differences in IgG1:IgG2 ratios observed here. IgG2 antibodies are generally associated with responses to carbohydrate antigens (Vidarsson et al., 2014), however, IgG2 has also been shown to recognize protein antigens. Indeed, two studies have demonstrated a link between IgG2 antibodies to HIV Gag proteins and natural control of HIV infection (Ngo-Giang-Huong et al., 2001; French et al., 2013). Nevertheless, a related study failed to detect differences in Gag-specific IgG2 levels between HIV controllers and chronic progressors (Banerjee et al., 2010). However, in contrast to the two studies, that used viral lysates in western blot assays, Banerjee et al. (2010) used recombinant HIV antigens in ELISAs, implying that conformational variations in HIV antigen could have affected IgG2-detection (Banerjee et al., 2010).Observations in this study that elevated levels of HIV-specific IgG1 and decreased IgG2 levels were associated with the G1m17-allele, support the hypothesis that allotypes could present useful genetic markers for the assessment of HIV-1 acquisition risk in vaccinated individuals. This concept was first tested on samples from the Step Study (Pandey et al., 2013), that failed to show protective efficacy. This phase IIb proof-of-concept study, designed to assess the efficacy of the MRK Ad5 gag/pol/nef HIV vaccine, was terminated prematurely on the grounds of futility. However, there was an observed increased risk of HIV-1 acquisition in the vaccine group when compared to the placebo group (Buchbinder et al., 2008; Gray et al., 2010). Pandey et al. (2013) went on to investigate whether this observed increased risk of HIV-1 acquisition could be linked to Ig allotype and revealed that the risk of HIV-1 acquisition was significantly increased in individuals positive for a combination of homozygous G1m17 and Km1 allotypes. Furthermore, the researchers found that subjects homozygous for FcyRIIIa (F-version) in the absence of G2m23 were more likely to become infected with HIV-1 (Pandey et al., 2013). The significance of these findings in terms of immune function are unclear given the vaccine approach was predicated on eliciting cellular responses designed to control viral replication rather than protective antibodies. Nevertheless, they serve to highlight the potential association of Ig allotype with HIV acquisition.In the present study, we applied a novel assay for probing the engagement of FcɣRIIa/ FcɣRIIIa dimers to investigate potential links between IgG1-allotypy and Fc-effector binding profiles (Wines et al., 2016). This has potential implications for HIV vaccine research since the significance of Fc-effector functions has been highlighted by a large phase III HIV-vaccine trial (RV144), in which HIV-specific ADCC was associated with enhanced protection against HIV-1 acquisition (Haynes et al., 2012; Kramski et al., 2015). A similar vaccine approach is now being pursued in the HVTN 702 trail evaluated in South Africa. The potential for IgG1 allotype to influence ADCC function is not without precedent, previously reported in prostate cancer where the capacity of NK cells to mediate ADCC against prostate cancer cells is influenced by interactions between different IgG1-allotypes and the corresponding FcɣRIIIa variants (Pandey and Namboodiri, 2014a).We determined the interactions of serum antibodies specific to different HIV-1 clades, with FcɣRIIa/ FcɣRIIIa-dimers and with respect to the IgG1-allotypes G1m1 and G1m3. Higher FcɣR-engagement by CN54gp140-specific serum antibodies was detected in G1m1-carriers as opposed to homozygous G1m3 carriers. Increased IgG1-titers that were associated with G1m1 are likely responsible for augmented crosslinking of FcɣRs given their higher affinity over IgG2. Interestingly, this trend of significantly increased FcɣR-engagement in G1m1-carriers by vaccine-induced serum antibodies was generally preserved against envelope proteins of different HIV-1 clades, implying that epistatic interactions between Fc-domains and FcɣR could play an important role in HIV-1 vaccines. This notion is supported by another study that investigated the distribution of FcɣR and Gm allotypes among HIV-1 controllers and non-controllers. The major finding was that among Caucasian Americans negative for the FcɣRIIa allele, Gm21-positive individuals were 7 times more likely to be HIV-1 controllers than non-carriers of Gm21, whereas this trend was not observed in the African American cohort (Deepe et al., 2012). It would be interesting to investigate at the sequence level, if similar links could be found for G1m3 and G1m1 allotypes in HIV-1 vaccine recipients. Although the results of this current study are preliminary, they suggest that individuals homozygous for G1m3 exhibit lower levels of Ag-specific IgG1 and lower FcɣR-engagement in response to HIV-vaccination. If FcɣR function is important for antibody mediated protection then these individuals would be less well protected than those homozygous for G1m1. These results have important implications for the two ongoing efficacy studies of HIV vaccines (HVTN 702 and 705) predicated on engagement of FcɣR mediated cellular functions including ADCC and ADCP. Our novel allotyping protocol provides new tools to determine the potential impact of IgG1 allotypes on vaccine efficacy.